Low Dead Volume Extraction Column Device

ABSTRACT

The invention provides extraction columns for the purification of an analyte (e.g., a biological macromolecule, such as a peptide, protein or nucleic acid) from a sample solution, as well as methods for making and using such columns. The invention is characterized by the use of low dead volume columns, which is achieved in part by the use of low pore volume frits (e.g., membrane screens) to contain a bed of extraction media in the column. Low dead volume facilitates the elution of the captured analyte into a very small volume of desorption solution, allowing for the preparation of low volume samples containing relatively high concentrations of analyte.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. patent applicationSer. No. 10/620,155 filed Jul. 14, 2003 which is incorporated byreference herein in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to a device and method for the capture ofanalytes by solid phase extraction with a column device and collectionof the analytes into a controlled volume of solvent. The analytes caninclude biomolecules, particularly biological macromolecules such asproteins and peptides. The device and method of this invention areparticularly useful in proteomics for sample preparation and analysiswith analytical technologies employing biochips, mass spectrometry andother instrumentation.

BACKGROUND OF THE INVENTION

Proteomics can be defined as the comprehensive study of proteins andtheir functional aspects. Proteins perform the work of the cell. Singleproteins can have many forms. The function of a protein depends on theform, interactions, and complexes of the protein. A deeper understandingof the biological functions of proteins is needed so that drugs can bedeveloped.

Protein sample processing is a complex problem within proteomics.Proteins can function individually or as complexes (groups of proteinsbound as a complex). Proteins cannot be amplified, as DNA is amplifiedwith polymerase chain reaction (PCR) methods. Proteins must be enrichedand purified before they can be analyzed. Protein processing methods andsystems must be flexible; more than a million possible proteins areexpressed. For analysis it is necessary to separate and concentrate theproteins of interest from many thousands of other proteins, whileselectively removing other materials that will interfere with theprotein analytical process including cellular material such as otherproteins, sugars, carbohydrates, lipids, DNA, RNA and salts.Reproducible recovery is needed and in most cases protein function mustbe retained during processing. Structural differences between forms mustbe preserved and final processing of samples must be easily integratedinto many different detection schemes, for example mass spectrometry,protein chips, and the like.

Solid phase extraction is one of the primary tools for preparing proteinsamples prior to analysis. The method purifies proteins according totheir identity, class type or structure, or function to prepare them foranalysis by mass spectrometry or other analytical methods.

The process of solid phase extraction uses an extraction phase in theform of a column or bed, and the sample may be either loaded onto thecolumn or added to a bulk solution to extraction beads. The extractionphase retains the sample protein, the extraction phase is washed toremove contaminants, and then the sample protein is removed with theextraction or recovery solvent.

Extraction columns are used to prepare the protein samples for analysis.Often very low amounts of proteins are expressed in a sample, and samplepreparation procedures are needed to isolate and recover the proteinbefore analysis.

The solid phase extraction of biomolecules such as nucleic acids andproteins is commonly performed by columns packed with a variety ofextraction phases.

The need for biomolecule extraction for proteins is increasing rapidly.Large numbers of samples need to be analyzed by a variety of techniquesto determine the function of proteins. Typical sample volume is 0.5 to 5mL or more on a typical column bed volume of 1 to 5 mL, requiring atypical desorption solvent volume of 2 to 10 mL.

There are a number of companies that have developed products whoseprinciple aim is the purification of certain proteins or protein classesby solid phase extraction. The intent of these products is thesimplification of proteomic analyses by providing a sample of only thoseproteins in which the investigator is interested. These products areoften packaged for a single use and disposal. Packed-bed columns operateat relatively low pressures, thus making them simple to operate in ahighly parallel and automated manner. Due to the very nature of aconventional packed-bed approach, it is limited with respect to reliablequantification and/or enrichment of sample. A packed-bed approach isextremely difficult to apply in a manner that is both cost-effective andreliable. It cannot be effectively applied to a microscale processlevel.

Moreover, packed columns have extensive carry-over from sample tosample, are expensive to manufacture, and may be difficult to multiplex(extract multiple samples simultaneously). Proteins may be irreversiblyadsorbed to the extraction phase or may be trapped by frits and other“dead zones” within the column making recovery of the proteinsincomplete.

Other drawbacks include losses of materials due to unswept volumesleading to low recoveries and irreproducibility of results; dilution ofmaterials due to large elution volumes applied in an attempt to minimizethese selfsame unswept volumes; depending on implementation,requirements often to adhere to a flow “directionality” introducinglimitations on full integration of sample processing; manufacturingdifficulties and costs for micro- or nanoscale volume systems; andporosity of construction materials used in commercially availablesystems that cause severe loss of biomaterials.

Spin columns and pipette tip columns are disposable column technologiescommonly used for processing samples. At present, most of these columnscontain filters or frits. Conventional frits, porous discs used tocontain the column beds, have significant dead volume. This leads tosignificant sample loss when very small sample volumes are separated.

One conventional method for making sample preparation devices involvesfirst inserting a precut porous plug obtained from, for example, afibrous glass or cellulose sheet, into the tip of a pipette. This isfollowed by the addition of loose particles and a second porous plug.The plugs serve to retain the particles in place in the pipette tip.However, the plugs also entrap excess liquid thereby creating dead spaceor volume (i.e., space not occupied by media or polymer that can lead topoor sample recovery, contamination such as by sample carry-over, etc.).

Current available methods are not well suited for the separation andrecovery of very small volumes in the low microliter range.

Also, since the volume of the filter is often as large as the volume ofthe micro volume sample itself, the extraction or separation process orchromatography process is adversely affected due to the large volume offilter material through which the sample must pass.

In addition, the adsorption of biomolecules can be a problem. Since theconcentration of biomolecules in micro volume samples is so small, theadsorption of biomolecules on the filter can result in significant lossof the total sample mass. The filter material may also absorb proteinsor biomolecules from the sample, resulting in lower than desirablesample recovery. Also, the filter material may behave differently indifferent elution media, subsequently interfering with both the qualityof the separation process and the volume of the sample retained.

Collecting samples in the 1 to 20 μL range is a critical need. At suchlow volumes, efficient sample handling is crucial to avoid loss.Conventional methods and devices for sample preparation are notpractical for handling the “microseparation” of such small samplevolumes.

Ultrafiltration can only effectively concentrate and desalt, and thusthe application of adsorption technology at this scale could offer anentirely new approach to micro-mass sample preparation.

However, these procedures cannot be used with extremely small liquiddelivery devices such as conventional pipette tips, as there is nopractical way to load either the plug or the particles to obtain amicro-adsorptive device that contains 20 milligrams or less ofadsorbent, the amount suitable for use with the aforementioned extremelysmall sample loads.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an embodiment of the invention where the extractioncolumn body is constructed from a tapered pipette tip.

FIG. 2 is an enlarged view of the extraction column of FIG. 1.

FIG. 3 depicts an embodiment of the invention where the extractioncolumn is constructed from two cylindrical members.

FIG. 4 depicts a syringe pump embodiment of the invention with acylindrical bed of solid phase media in the tip.

FIG. 5 is an enlarged view of the extraction column element of thesyringe pump embodiment of FIG. 4.

FIGS. 6-10 show successive stages in the construction of the embodimentdepicted in FIGS. 1 and 2.

FIG. 11 depicts an embodiment of the invention with a straightconnection configuration as described in Example 8.

FIG. 12 depicts an embodiment of the invention with an end cap andretainer ring configuration as described in Example 9.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

This invention is used for the capture of analytes by solid phaseextraction with a column device and collection of the analytes into acontrolled volume of solvent. This invention is useful for analytesincluding biomolecules and is compatible with requirements for samplepreparation and analysis by analytical technology—especially biochipsand mass spectrometry.

The invention is characterized by the use of extraction columns havinglow dead volumes. This is achieved in part by the use of a low volumefrit or frits to contain a bed of extraction media in an extractionmedia chamber positioned in the column. Low dead volume facilitates theelution of the captured analyte into a very small volume of desorptionsolution, allowing for the preparation of low volume samples containingrelatively high concentrations of analyte. Low volume, highconcentration solutions particularly useful with regard to proteinpreparations for analysis by techniques such as mass spectrometery andprotein chips.

I. Terminology

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific embodimentsdescribed herein. It is also to be understood that the terminology usedherein for the purpose of describing particular embodiments is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to polymer bearing a protected carbonyl would include apolymer bearing two or more protected carbonyls, and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, specific examples ofappropriate materials and methods are described herein.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

The term “bed volume” as used herein is defined as the volume of a bedof extraction media in an extraction column. Depending on how denselythe bed is packed, the volume of the extraction media in the column bedis typically about half to one third of the total bed volume; wellpacked beds have less space between the beads and hence generally havelower interstital volumes.

The term “interstitial volume” of the bed refers to the volume of thebed of extraction media that is accessible to solvent, e.g., aqueoussample solutions, wash solutions and desorption solvents. For example,in the case where the extraction media is a chromatography bead (e.g.,agarose or sepharose), the interstitial volume of the bed constitutesthe solvent accessible volume between the beads, as well as any solventaccessible internal regions of the bead, e.g., solvent accessible pores.The interstitial volume of the bed represents the minimum volume ofliquid required to saturate the column bed.

The term “dead volume” as used herein with respect to a column isdefined as the interstitial volume of the extraction bed, tubes,membrane or frits, and passageways in a column. In the device of thisinvention with gel-type extraction media and the pore volume of thefrits. Since the bottom frit of the column directly contacts the sample,wash, and elution liquids, minimal tubing or passageway dead volume ispresent in this device.

The term “elution volume” as used herein is defined as the volume ofdesorption or elution liquid into which the analytes are desorbed andcollected. The terms “desorption solvent,” elution liquid” and the likeare used interchangeably herein.

The term “enrichment factor” as used herein is defined as the ratio ofthe sample volume divided by the elution volume, assuming that there isno contribution of liquid coming from the dead volume. To the extentthat the dead volume either dilutes the analytes or prevents completeadsorption, the enrichment factor is reduced.

The terms “extraction column” and “extraction tip” as used herein aredefined as a column device used in combination with a pump, the columndevice containing a bed of solid phase extraction material, i.e.,extraction media.

The term “frit” as used herein are defined as porous material forholding the extraction media in place in a column. An extraction mediachamber is typically defined by a top and bottom frit positioned in anextraction column. In preferred embodiments of the invention the frit isthin, and has a low pore volume, e.g., a membrane screen.

The term “gel-type packing material” as used herein is defined asnon-porous or micro-porous beads such as agarose or sepharose beads, thebeads containing a functional group or having a surface that bindsselectively with the analyte of interest.

The term “lower column body” as used herein is defined as the column bedand bottom membrane screen of a column.

The term “membrane screen” as used herein is defined as a woven ornon-woven fabric or screen for holding the column packing in place inthe column bed, the membranes having a low dead volume. The membranesare of sufficient strength to withstand packing and use of the columnbed and of sufficient porosity to allow passage of liquids through thecolumn bed. The membrane is thin enough so that it can be sealed aroundthe perimeter or circumference of the membrane screen so that theliquids flow through the screen.

The term “sample volume”, as used herein is defined as the volume of theliquid of the original sample solution from which the analytes areseparated or purified.

The term “upper column body”, as used herein is defined as the chamberand top membrane screen of a column.

The term “biomolecule” as used herein refers to biomolecule derived froma biological system. The term includes biological macromolecules, suchas a proteins, peptides, and nucleic acids.

The term “protein chip” is defined as a small plate or surface uponwhich an array of separated, discrete protein samples are to bedeposited or have been deposited. These protein samples are typicallysmall and are sometimes referred to as “dots.” In general, a chipbearing an array of discrete proteins is designed to be contacted with asample having one or more biomolecules which may or may not have thecapability of binding to the surface of one or more of the dots, and theoccurrence or absence of such binding on each dot is subsequentlydetermined. A reference that describes the general types and functionsof protein chips is Gavin MacBeath, Nature Genetics Supplement, 32:526(2002).

II. Low Dead Volume Extraction Columns

Column Body

The column body is a tube having two open ends connected by an openchannel. The tube can be in any shape, including but not limited tocylindrical or frustoconical, and of any dimensions consistent with thefunction of the column as described herein. In certain embodiments ofthe invention the column body takes the form of a pipette tip, asyringe, a luer adapter or similar tubular bodies.

One of the open ends of the column, sometimes referred to herein as theopen upper end of the column, is adapted for attachment to a pump. Insome embodiments of the invention the upper open end is operativelyattached to a pump, whereby the pump can be used for aspirating a fluidinto the extraction column through the other open end of the column, andoptionally for discharging fluid out through the open lower end of thecolumn. Thus, it is a feature of the present invention that fluid entersand exits the extraction column through the same open end of the column.This is in contradistinction with the operation of some extractioncolumns, where fluid enters the column through one open end and exitsthrough the other end after traveling through an extraction media, i.e.,similar to conventional column chromatography. The fluid can be aliquid, such as a sample solution, wash solution or desorption solvent.

The column body can be can be composed of any material that issufficiently non-porous that it can retain fluid and that is compatiblewith the solutions, media, pumps and analytes used. A material should beemployed that does not substantially react with substances it willcontact during use of the extraction column, e.g., the sample solutions,the analyte of interest, the extraction media and desorption solvent. Awide range of suitable materials are available and known to one of skillin the art, and the choice is one of design. Various plastics make idealcolumn body materials, but other materials such as glass, ceramics ormetals could be used in some embodiments of the invention. Some examplesof materials include polysulfone, polypropylene, polyethylene,polyethyleneterephthalate, polyethersulfone, polytetrafluoroethylene,cellulose acetate, cellulose acetate butyrate, acrylonitrile PVCcopolymer, polystyrene, polystyrene/acrylonitrile copolymer,polyvinylidene fluoride, glass, metal, silica, and combinations of theabove listed materials.

Some specific examples of suitable column bodies are provided in theExamples.

Extraction Media

The extraction media used in the column is preferably a form ofwater-insoluble particle (e.g., a porous or non-porous bead) that has anaffinity for an analyte of interest. Typically the analyte of interestis a protein, peptide or nucleic acid. The extraction processes can beaffinity, reverse phase, normal phase, ion exchange, hydrophobicinteraction chromatography, or hydrophilic interaction chromatographyagents.

The bed volume of the extraction media used in the extraction columns ofthe invention is typically small, preferably in the range of 0.5-100 μL,more preferably in the range of 1-50 μL, and still more preferably inthe range of 2-25 μL. The low bed volume results in a low interstitialvolume of the bed, contributing to the low dead volume of the column,thereby facilitating the recovery of the analyte in a small volume ofdesorption solvent.

The low bed volumes employed in certain embodiments allow for the use ofrelatively small amounts of extraction media, e.g., soft, gel-typebeads. For example, some embodiments of the invention employ a bed ofextraction media having a dry weight of less than 10 mg (e.g., in therange of 0.1-10 mg, 0.5-10 mg, 1-10 mg or 2-10 mg), less than 2 ms(e.g., in the range of 0.1-2 mg, 0.5-2 mg or 1-2 mg), or less than 1 mg(e.g., in the range of 0.1-1 mg or 0.5-1 mg).

Many of the extraction media types suitable for use in the invention areselected from a variety of classes of chromatography media. It has beenfound that many of these chromatography media types and the associatedchemistries are suited for use as solid phase extraction media in thedevices methods of this invention.

Thus, examples of suitable extraction media include agarose-basedmaterials, sepharose-based materials, polystyrene/divinylbenzenecopolymers, poly methylmethacrylate, protein G beads (e.g., for IgGprotein purification), MEP Hypercel™ beads (e.g., for IgG proteinpurification), affinity phase beads (e.g., for protein purification),ion exchange phase beads (e.g., for protein purification), hydrophobicinteraction beads (e.g., for protein purification), reverse phase beads(e.g., for nucleic acid or protein purification), and beads having anaffinity for molecules analyzed by label-free detection. Silica beadsare also suitable.

Soft gel-type beads, such as agarose and sepharose based beads, arefound to work surprisingly well in columns and methods of thisinvention. In conventional chromatography fast flow rates can result inbead compression, which results in increased back pressure and adverselyimpacts the ability to use these gels with faster flow rates. In thepresent invention relatively small bed volumes are used, and it appearsthat this allows for the use of high flow rates with a minimal amount ofbead compression and the problem attendant with such compression.

Affinity extractions use a technique in which a biospecific adsorbent isprepared by coupling a specific ligand (such as an enzyme, antigen, orhormone) for the analyte, (e.g., macromolecule) of interest to a solidsupport. This immobilized ligand will interact selectively withmolecules that can bind to it. Molecules that will not bind eluteunretained. The interaction is selective and reversible. The referenceslisted below show examples of the types of affinity groups that can beemployed in the practice of this invention are hereby incorporated byreference herein in their entireties. Antibody Purification Handbook,Amersham Biosciences, Edition AB, 18-1037-46 (2002); ProteinPurification Handbook, Amersham Biosciences, Edition AC, 18-1132-29(2001); Affinity Chromatography Principles and Methods, AmershamPharmacia Biotech, Edition AC, 18-1022-29 (2001); The RecombinantProtein Handbook, Amersham Pharmacia Biotech, Edition AB, 18-1142-75(2002); and Protein Purification: Principles, High Resolution Methods,and Applications, Jan-Christen Janson (Editor), Lars G. Ryden (Editor),Wiley, John & Sons, Incorporated (1989).

Examples of suitable affinity binding agents are summarized in Table I,wherein the affinity agents are from one or more of the followinginteraction categories:

-   -   1. Chelating metal—ligand interaction    -   2. Protein—Protein interaction    -   3. Organic molecule or moiety—Protein interaction    -   4. Sugar—Protein interaction    -   5. Nucleic acid—Protein interaction    -   6. Nucleic acid—nucleic acid interaction

TABLE I Examples of Affinity molecule or moiety fixed at Interactionsurface Captured biomolecule Category Ni-NTA His-tagged protein 1 Ni-NTAHis-tagged protein within a 1, 2 multi-protein complex Fe-IDAPhosphopeptides, 1 phosphoproteins Fe-IDA Phosphopeptides or 1, 2phosphoproteins within a multi-protein complex Antibody or otherProteins Protein antigen 2 Antibody or other Proteins Smallmolecule-tagged 3 protein Antibody or other Proteins Smallmolecule-tagged 2, 3 protein within a multi- protein complex Antibody orother Proteins Protein antigen within a 2 multi-protein complex Antibodyor other Proteins Epitope-tagged protein 2 Antibody or other ProteinsEpitope-tagged protein 2 within a multi-protein complex Protein A,Protein G or Antibody 2 Protein L Protein A, Protein G or Antibody 2Protein L ATP or ATP analogs; 5′- Kinases, phosphatases 3 AMP (proteinsthat requires ATP for proper function) ATP or ATP analogs; 5′- Kinase,phosphatases 2, 3 AMP within multi-protein complexes Cibacron 3G Albumin3 Heparin DNA-binding protein 4 Heparin DNA-binding proteins 2, 4 withina multi-protein complex Lectin Glycopeptide or 4 glycoprotein LectinGlycopeptide or 2, 4 glycoprotein within a multi-protein complex ssDNAor dsDNA DNA-binding protein 5 ssDNA or dsDNA DNA-binding protein 2, 5within a multi-protein complex ssDNA Complementary ssDNA 6 ssDNAComplementary RNA 6 Streptavidin/Avidin Biotinylated peptides 3 (ICAT)Streptavidin/Avidin Biotinylated engineered tag 3 fused to a protein(see avidity.com) Streptavidin/Avidin Biotinylated protein 3Streptavidin/Avidin Biotinylated protein within 2, 3 a multi-proteincomplex Streptavidin/Avidin Biotinylated engineered tag 2, 3 fused to aprotein within a multi-protein complex Streptavidin/Avidin Biotinylatednucleic acid 3 Streptavidin/Avidin Biotinylated nucleic acid 2, 3 boundto a protein or multi- protein complex Streptavidin/Avidin Biotinylatednucleic acid 3, 6 bound to a complementary nucleic acid

In one aspect of the invention an extraction media is used that containsa surface functionality that has an affinity for a protein fusion tagused for the purification of recombinant proteins. A wide variety offusion tags and corresponding affinity groups are available and can beused in the practice of the invention.

One of the most common fusion tags is the so-called “His” tag, which iscomprised of a series of consecutive histidine residues, e.g., two, fouror six consecutive histidine residues. There are a number ofmetal-chelate groups that can be attached to the surface of anextraction media for purification of “His-tagged proteins, includingmetal-IDA (IDA: iminodiacetate), metal-NTA (NTA: nitrilotriacetate), andmetal-CMA (CMA: carboxymethylated aspartate), where the metal istypically selected from nickel, copper, iron, zinc and cobalt. Thetrapped fusion protein is eluted by disrupting the histidine-metalcoordination by some suitable salt such as imidazole or ethylene diaminetetra acetic acid (EDTA).

There are other affinity groups available for purifying recombinantproteins through their fusion tags, and these groups can be attached toan extraction media for use in the invention. Antibodies can be used forpurification through any peptide sequence (a common one is the FLAGtag); avidin (monomeric or multimeric) can be used for purifying apeptide sequence that is selectively biotinylated within the expressionsystem; calmodulin charged with calcium can be used for purifying apeptide sequence that is often referred to as a “calmodulin bindingpeptide” (or, CBP), where elution is performed by removing the calciumwith ethylene glycol tetra acetic acid (EGTA); glutathione can be usedfor purifying a fusion protein that carries the glutathioneS-transferase protein (GST), where the GST is often cleaved off with aspecific protease; amylose can be used for purifying a fusion proteinthat carries the maltose binding protein (MBP), where the MBP is oftencleaved off with a specific protease; cellulose can be used forpurifying a fusion protein that carries a peptide that is referred to asthe cellulose-binding domain tag, followed by elution with ethyleneglycol; S-protein (derived from ribonuclease A) can be used forpurifying a fusion protein that carries a peptide with specific affinityfor S-protein, where the peptide can be cleaved off with a specificprotease.

It is also possible to create an affinity surface that has thebis-arsenical fluorescein dye FlAsH. For example, a FlAsH dye can beused for purifying a fusion protein that carries the peptide sequencetag CCxxCC (where xx is any amino acid, such as RE). The protein is theneluted with 1,4-dithiothreitol, or DTT.

In one aspect the invention is used for purification of antibodies.Antibodies are frequently purified on the basis of highly conservedstructural characteristics. For example, it is possible to pack columnswith extraction media containing Protein A, Protein G, or Protein A/Gfusions to purify IgG antibodies through their Fc region (with loweraffinity for the Fab antibody fragment region in the case of Protein G).These are often eluted by using low pH 2.5. It is also possible topurify IgG antibodies through their Fab antibody fragment region,provided their light chain is a kappa light chain. This is achieved byusing a surface of Protein L.

In one aspect the extraction media comprises small molecule ligands thatare capable of achieving separations on the basis of hydrophobic chargeinteractions. Ligands such as 4-mercapto-ethyl-pyridine and2-mercaptopyridine are capable of trapping antibodies such as IgGs,which are eluted by changes to low pH much milder than in the case ofProtein A or Protein G. For example, elution is accomplished with4-mercapto-ethyl-pyridine at pH 4 (as opposed to pH 2.5 for the ProteinA and Protein G).

In addition, other antibodies can be used for purification ofantibodies. For example, it is possible to use an extraction mediacomprising an immobilized antibody for the purification of IgE (with ananti-IgE surface), the purification of IgM (with an anti-IgM surface),the purification of IgA (with an anti-IgA surface), the purification ofIgD (with an anti-IgD surface), as well as the purification of IgG (withan anti-IgG surface).

Extraction columns of the invention can be used for purification ofphosphopeptides and phosphoproteins by the inclusion of an appropriateaffinity group on the extraction media. One alternative is to exploitthe natural interaction between phosphate groups and metal ions.Therefore, phosphopeptides and phosphoproteins can be purified onmetal-chelate surfaces made from IDA, NTA, or CMA.

It is also possible to purify these phosphopeptides and phosphoproteinswith immobilized antibodies. For example, it is possible to useantibodies on the packing material that are specific to phosphotyrosineresidues, as well as phosphoserine and phosphothreonine residues. It isalso possible to use antibodies that are bind to specific phosphorylatedsites within a protein, such as specifically-binding phosphorylatedtyrosine within a specific kinase. These antibodies are often referredto as phosphorylation site-specific antibodies (PSSAs). Once adsorbedthe trapped phosphoprotein and phosphopeptides can be eluted at low pH.

Yet another approach to the purification of phosphopeptides andphosphoproteins involves the derivitization of the phosphate group suchthat biotin is attached to it. This biotinylated phosphoprotein orphosphopeptide can be purified using an avidin-derivatized extractionmedia, wherein the avidin can be monomeric or multimeric.

In some embodiments of the invention an extraction column is used forthe purification of protein complexes. One embodiment involves the useof a recombinant “bait” protein that will form complexes with itsnatural interaction partners. These multiprotein complexes are thenpurified through a fusion tag that is attached to the “bait.” Thesetagged “bait” proteins can be purified through groups incorporated intothe extraction media such as metal-chelate groups, antibodies,calmodulin, or any of the other surface groups described above for thepurification of recombinant proteins.

It is also possible to purify “native” (i.e. non-recombinant) proteincomplexes without having to purify through a fusion tag. This isachieved by immobilizing an antibody for one of the proteins within themultiprotein complex. This process is often referred to as“co-immunoprecipitation.” The multiprotein complexes can be eluted withlow pH.

Extraction columns of the invention can be used to purify entire classesof proteins on the basis of highly conserved motifs within theirstructure, whereby an affinity ligand attached to the packing reversiblybinds to the conserved motif. For example, it is possible to immobilizeparticular nucleotides on the extraction media. Examples include, butare not limited to, adenosine 5′-triphosphate (ATP), adenosine5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), nicotinamideadenine dinucleotide (NAD), or nicotinamide adenine dinucleotidephosphate (NADP). These nucleotides can be used for the purification ofenzymes that are dependent upon these nucleotides such as kinases,phosphatases, heat shock proteins and dehydrogenases, to name a few.

There are other affinity groups that can be incorporated into theextraction media for purification of protein classes. Lectins can beused for the purification of glycoproteins. Concanavilin A (Con A) andlentil lectin can be used for the purification of glycoproteins andmembrane proteins, and wheat germ lectin can be used for thepurification of glycoproteins and cells (especially T-cell lymphocytes).Though it is not a lectin, the small molecule phenylboronic acid canalso be incorporated into the extraction media and used for purificationof glycoproteins.

It is also possible to incorporate heparin into the extraction media,which is useful for the purification of DNA-binding proteins (e.g. RNApolymerase I, II and III, DNA polymerase, DNA ligase). In addition,immobilized heparin can be used for purification of various coagulationproteins (e.g. antithrombin III, Factor VII, Factor IX, Factor XI,Factor XII and XIIa, thrombin), other plasma proteins (e.g. properdin,BetaIH, Fibronectin, Lipses), lipoproteins (e.g. VLDL, LDL, VLDLapoprotein, HOLP, to name a few), and other proteins (platelet factor 4,hepatitis B surface antigen, hyaluronidase). These types of proteins areoften blood and/or plasma borne. Since there are many efforts afoot torapidly profile the levels of these types of proteins by technologiessuch as protein chips, the performance of these chips will be enhancedby performing an initial purification and enrichment of the targetsprior to protein chip analysis.

It is also possible to use extraction media with protein interactiondomains for purification of those proteins that are meant to interactwith that domain. One interaction domain that can be used is theSrc-homology 2 (SH2) domain that binds to specificphophotyrosine-containing peptide motifs within various proteins. TheSH2 domain has previously been immobilized on a resin and used as anaffinity reagent for performing affinity chromatography/massspectrometry experiments for investigating in vitro phosphorylation ofepidermal growth factor receptor (EGFR) (see Christian Lombardo, et al.,Biochemistry, 34:16456 (1995)). Other than the SH2 domain, other proteininteraction domains can be used for the purposes of purifying thoseproteins that possess their recognition domains. Many of these proteininteraction domains have been described (see Tony Pawson, ProteinInteraction Domains, Cell Signaling Technology Catalog, 264-279 (2002))for additional examples of these protein interaction domains).

Benzamidine is another example of a class-specific affinity ligand,which can be incorporated into the extraction media for purification ofserine proteases. The dye ligand Procion Red HE-3B can be used for thepurification of dehydrogenases, reductases and interferon, to name afew.

Reversed-phase chromatography media can also function as an extractionmedia in certain embodiments of the invention. In reversed-phasechromatography, an aqueous/organic solvent mixture is commonly used asthe mobile phase, and a high-surface-area nonpolar solid is employed asthe stationary phase. The latter can be an alkyl-bonded silica packing,e.g., with C₈ or C₁₈ groups covering the silica surface. The basis ofsolute retention in reversed-phase chromatography is still somewhatcontroversial; some workers favor an adsorption, while others believethat the solute partitions into the nonpolar stationary phase. Probablyboth processes are important for many samples. Competition betweensolute and mobile-phase molecules exists for a place on thestationary-phase surface. That is, an adsorbed molecule will displacesome number of previously adsorbed molecules (Chromatography, 5^(th)edition, PART A: FUNDAMENTALS AND TECHNIQUES, editor: E. Heftmann,Elsevier Science Publishing Company, New York, pp A25 (1992)). The nearuniversal application of reversed-phase chromatography stems from thefact that virtually all organic molecules have hydrophobic regions intheir structure and are capable of interacting with the stationaryphase. Since the mobile phase is polar and generally contains water, themethod is ideally suited to the separation of polar molecules which areeither insoluble in organic solvents or bind too strongly to inorganicoxide adsorbents for normal elution. Reversed-phase chromatographyemploying acidic, low ionic strength eluents has become a widelyestablished technique for the purification and structural elucidation ofproteins. However, the structure of biopolymers is very sensitive tomobile phase composition, pH and the presence of complexing specieswhich can result in anomalous retention and even denaturing of proteins.A general characteristic of reversed-phase systems is that a decrease inpolarity of the mobile phase, that is increasing the volume fraction oforganic solvent in an aqueous organic mobile phase, leads to a decreasein retention; a reversal of the general trends observed in liquid-solidchromatography or normal phase chromatography. It is also generallyobserved for reversed-phase chromatography that for members of ahomologous or oligomous series, the logarithm of the solute capacityfactor is a linear function of the number of methylene groups or repeatunits of the oligomeric structure (ADVANCED CHROMATOGRAPHIC ANDELECTROMIGRATION METHODS IN BIOSCIENCES, editor: Z. Deyl, ElsevierScience BV, Amsterdam, The Netherlands, pp 528 (1998); CHROMATOGRAPHYTODAY, Colin F. Poole and Salwa K. Poole, and Elsevier SciencePublishing Company, New York, pp 394 (1991)).

The references listed below show different types of surfaces used forreverse phase separations and are hereby incorporated by referenceherein in their entireties: CHROMATOGRAPHY, 5^(th) edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A25 (1992); ADVANCED CHROMATOGRAPHICAND ELECTROMIGRATION METHODS IN BIOSCIENCES, editor: Z. Deyl, ElsevierScience BV, Amsterdam, The Netherlands, pp 528 (1998); CHROMATOGRAPHYTODAY, Colin F. Poole and Salwa K. Poole, and Elsevier SciencePublishing Company, New York, pp 394 (1991).

Ion-pair chromatography media can also function as an extraction mediain certain embodiments of the invention. In ion-pair chromatography, thecolumn packing is usually the same as in reversed-phase chromatography;e.g., a C₈ or C₁₈ silica. The mobile phase is likewise similar to thatused in reverse phase chromatography: an aqueous/organic solvent mixturecontaining a buffer plus a so-called ion-pair reagent. The ion-pairreagent will be positively charged for the retention and separation ofsample anions and negatively charged for the retention of samplecations. Typical examples of ion-pair reagents are hexane sulfonate andtetrabutylammonium. The basis of retention in ion-pair chromatography isstill controversial, two different processes being possible: (a)adsorption of ion pairs or (b) formation of an in situ ion exchanger.Although these two processes appear somewhat different, they lead toquite similar predictions of retention as a function of experimentalconditions. Retention in ion-pair chromatography can be continuouslyvaried from a reversed-phase process to an ion-exchange process. Thiscapability provides a number of practical advantages. For example,variation of the mobile phase composition allows a considerable controlover the retention of individual sample ions. This can be used toseparate particularly difficult samples, e.g., mixtures of anionic,cationic, and/or neutral molecules (CHROMATOGRAPHY, 5^(th) Edition, PartA: Fundamentals And Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A28 (1992)).

The references listed below show different types of groups used forion-pair chromatography and are hereby incorporated by reference hereinin their entireties: Reference: CHROMATOGRAPHY, 5^(th) Edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A28 (1992); and CHROMATOGRAPHY TODAY,Colin F. Poole and Salwa K. Poole, Elsevier Science Publishing Company,New York, pp 411 (1991).

Normal phase chromatography media can also function as an extractionmedia in certain embodiments of the invention. In normal phasechromatography, the stationary phase is a high-surface-area polaradsorbent, e.g., silica or a bonded silica with polar surface groups.The mobile phase (a mixture of organic solvents) is less polar than thestationary phase. Consequently, more polar solutes are preferentiallyretained; there is often little difference in the retention of differenthomologues or a particular compound class. This has led to the use ofnormal phase chromatography for so-called compound-class (group-type)separations, where, e.g., alcohols are separated as a group frommonoesters and other compound classes. The basis of normal phasechromatography retention is an adsorption/displacement process. Anotherfeature of normal phase chromatography retention is the so-calledlocalization of adsorbed solute and mobile-phase molecules on thestationary-phase surface. Localization refers to the formation ofdiscrete bonds (by dipole/dipole or hydrogen-bonding interactions)between polar sites on the adsorbent and polar substituents in thesolute molecule. Localization, in turn, confers a high degree ofspecificity to the interaction of solute isomers with the adsorbentsurface, leading to typically better separations of isomers by normalphase chromatography than by other chromatographic methods(CHROMATOGRAPHY, 5^(th) edition, Part A: Fundamentals and Techniques,editor: E. Heftmann, Elsevier Science Publishing Company, New York, ppA27 (1992)).

The references listed below show different types of affinity groups usedfor normal phase chromatography and are hereby incorporated by referenceherein in their entireties: CHROMATOGRAPHY, 5^(th) edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A27 (1992); and CHROMATOGRAPHY TODAY,Colin F. Poole and Salwa K. Poole, Elsevier Science Publishing Company,New York, pp 375 (1991).

Ion Exchange chromatography media can also function as an extractionmedia in certain embodiments of the invention. Ion Exchange (IEX) is amode of chromatography in which ionic substances are separated oncationic or anionic sites of the packing. The surface in ion exchange isusually an organic matrix which is substituted with ionic groups, e.g.,sulfonate or trimethylammonium. The mobile phase typically consists ofwater plus buffer and/or salt. The retention of a solute ion occurs viaion exchange with a mobile phase ion or similar (positive or negative)charge. Ion exchange chromatography is often applied to the separationof acidic or basic samples, whose charge varies with pH. In the simplecase of solute molecules bearing a single acidic or basic group, thesolute will be present as some mixture of charged and neutral species.The fraction of solute molecules that are ionized then determinesretention. In the case of ion exchange, the retention of the unchargedspecies can be ignored (CHROMATOGRAPHY, 5^(th) Edition, Part A:Fundamentals and Techniques, editor: E. Heftmann, Elsevier SciencePublishing Company, New York, pp A28 (1992)). Ion exchangechromatography is one of the oldest and most traditional techniques forseparating complex mixtures of proteins. The references listed belowshow different types of groups and surfaces used for ion exchangechromatography and are hereby incorporated by reference herein in theirentireties; CHROMATOGRAPHY, 5^(th) Edition, Part A: Fundamentals andTechniques, editor: E. Heftmann, Elsevier Science Publishing Company,New York, pp A28 (1992); CHROMATOGRAPHY TODAY, Colin F. Poole and SalwaK. Poole, Elsevier Science Publishing Company, New York, pp 422 (1991);and ADVANCED CHROMATOGRAPHIC AND ELECTROMIGRATION METHODS INBIOSCIENCES, editor: Z. Deyl, Elsevier Science BV, Amsterdam, TheNetherlands, pp 540 (1998).

Hydrophobic Interaction Chromatography media can also function as anextraction media in certain embodiments of the invention. HydrophobicInteraction Chromatography is widely used for the separation andpurification of proteins. During separation, proteins are induced tobind to a weakly hydrophobic stationary phase using a buffered mobilephase of high ionic strength and then selectively desorbed during adecreasing salt concentration gradient. Proteins are usually separatedin hydrophobic interaction chromatography according to their degree ofhydrophobicity, much as in reversed-phase chromatography, but because ofthe gentler nature of the separation mechanism, there is a greaterprobability that they will elute with their conformational structure(biological activity) intact. In reversed-phase chromatography, proteinsunfold on the bonded phase surface as a consequence of the highinterfacial tension existing between the mobile and the bondedstationary phases. These conditions are minimized in hydrophobicinteraction chromatography by using stationary phases of lowerhydrophobicity together with totally aqueous mobile phases, in general,since solvent strength is controlled by varying ionic strength ratherthan by increasing the volume fraction of an organic modifier. Retentionand selectivity in hydrophobic interaction chromatography dependsubstantially on the type of stationary phase. Retention increases formore hydrophobic ligands and with it the possibility of denaturingcertain proteins. Some proteins are only satisfactorily handled onhydrophilic stationary phases. The ligand density and structure as wellas the hydrophobicity of the stationary phase are the primary stationaryphase variables that should be optimized for the separation ofindividual proteins. Mobile phase parameters that have to be optimizedare the salt concentration, salt type, slope of the salt gradient, pH,addition of surfactant or organic modifier and temperature. In theabsence of specific binding of the salt to the protein molecule and atrelatively high salt concentration in the mobile phase, retentionincreases linearly with the salt molality and at constant saltconcentration with the molal surface tension increment of the salt usedin the aqueous mobile phase.

The reference listed below shows different types of groups and surfacesused for hydrophobic interactions and is hereby incorporated byreference herein in its entirety: CHROMATOGRAPHY TODAY, Colin F. Pooleand Salwa K. Poole, Elsevier Science Publishing Company, New York, 402(1991).

Frits

Columns of the invention employ frits having a low pore volume, whichcontributes to the low dead volume of the columns. The frits of theinvention are porous, since it is necessary for fluid to be able to passthrough the frit. The frit should have sufficient structural strength sothat frit integrity can contain the extraction media in the column. Itis desirable that the frit have little or no affinity for chemicals withwhich it will come into contact during the extraction process,particularly the analyte of interest. In many embodiments of theinvention the analyte of interest is a biomolecule, particularly abiological macromolecule. Thus in many embodiments of the invention itdesirable to use a frit that has a minimal tendency to bind or otherwiseinteract with biological macromolecules, particularly proteins, peptidesand nucleic acids.

Frits of various pores sizes and pore densities may be used provided thefree flow of liquid is possible and the beads are held in place withinthe extraction media bed.

One frit, i.e., a lower frit, is bonded to and extends across the openchannel of the column body. A second frit is bonded to and extendsacross the open channel between the bottom frit and the open upper endof the column body.

The top frit, bottom frit and channel surface define an extraction mediachamber wherein a bed of extraction media is positioned. The fritsshould be securely attached to the column body and extend across theopening and/or open end so as to completely occlude the channel, therebysubstantially confining the bed of extraction media inside theextraction media chamber. In certain embodiments of the invention thebed of extraction media occupies at least 80% of the volume of theextraction media chamber, more preferably 90%, 95%, 99%, orsubstantially 100% of the volume. In some embodiments the invention thespace between the extraction media bed and the upper and lower frits isnegligible, i.e., the frits are in substantial contact with upper andlower surfaces of the extraction media bed, holding a well-packed bed ofextraction media securely in place.

In some embodiments of the invention the bottom frit is located at theopen lower end of the column body. This configuration is shown in thefigures and exemplified in the Examples, but is not required, i.e., insome embodiments the bottom frit is located at some distance up thecolumn body from the open lower end. However, in view of the importanceof minimizing dead volume in the column it is desirable that the lowerfrit and extraction media chamber be located at or near the lower end.In some cases this can mean that the bottom frit is attached to the faceof the open lower end, as shown in FIGS. 1-10. However, in some casesthere can be some portion of the lower end extending beyond the bottomfrit, as exemplified by the embodiment depicted in FIG. 11. For thepurposes of this invention, so long as the length as this extension issuch that it does not substantially introduce dead volume into theextraction column or otherwise adversely impact the function of thecolumn, the bottom frit is considered to be located at the lower end ofthe column body. In some embodiments of the invention the volume definedby the bottom frit, channel surface, and the face of the open lower end(i.e., the channel volume below the bottom frit) is less than the volumeof the extraction media chamber, or less than 10% of the volume of theextraction media chamber, or less than 1% of the volume of theextraction media chamber.

The frits used in the invention are characterized by having a low porevolume. Some embodiments of the invention employ frits having porevolumes of less than 1 microliter (e.g., in the range of 0.015-1microliter, 0.03-1 microliter, 0.1-1 microliter or 0.5-1 microliter),preferably less than 0.5 microliter (e.g., in the range of 0.015-0.5microliter, 0.03-0.5 microliter or 0.1-0.5 microliter), less than 0.1microliter (e.g., in the range of 0.015-0.1 microliter or 0.03-0.1microliter) or less than 0.03 microliters (e.g., in the range of0.015-0.03 microliter).

Frits of the invention preferably have pore openings or mesh openings ofa size in the range of about 5-100 μm, more preferably 10-100 μm, andstill more preferably 15-50 μm, e.g, about 43 μm. The performance of thecolumn is typically enhanced by the use of frits having pore or meshopenings sufficiently large so as to minimize the resistance to flow.The use of membrane screens as described herein typically provide thislow resistance to flow and hence better flow rates, reduced backpressure and minimal distortion of the bed of extraction media. The poreor mesh openings of course should not be so large that they are unableto adequately contain the extraction media in the chamber.

The frits used in the practice of the invention are characterized byhaving a low pore volume relative to the interstitial volume of the bedof extraction media contained by the frit. Thus, in certain embodimentsof the invention the frit pore volume is equal to 10% or less of theinterstitial volume of the bed of extraction media (e.g., in the range0.1-10%, 0.25-10%, 1-10% or 5-10% of the interstitial volume), morepreferably 5% or less of the interstitial volume of the bed ofextraction media (e.g., in the range 0.1-5%, 0.25-5% or 1-5% of theinterstitial volume), and still more preferably 1% or less of theinterstitial volume of the bed of extraction media (e.g., in the range0.1-1% or 0.25-0% of the interstitial volume).

The pore density will allow flow of the liquid through the membrane andis preferably 10% and higher to increase the flow rate that is possibleand to reduce the time needed to process the sample.

Some embodiments of the invention employ a thin frit, preferably lessthan 350 μm in thickness (e.g., in the range of 20-350 μm, 40-350 μm, or50-350 μm), more preferably less than 200 μm in thickness (e.g., in therange of 20-200 μm, 40-200 μm, or 50-200 μm), more preferably less than100 μm in thickness (e.g., in the range of 20-100 μm, 40-100 μm, or50-100 μm), and most preferably less than 75 μm in thickness (e.g., inthe range of 20-75 μm, 40-75 μMm, or 50-75 μm).

Some embodiments of the invention employ a membrane screen as the frit.The membrane screen should be strong enough to not only contain theextraction media in the column bed, but also to avoid becoming detachedor punctured during the actual packing of the media into the column bed.Membranes can be fragile, and in some embodiments must be contained in aframework to maintain their integrity during use. However, it isdesirable to use a membrane of sufficient strength such that it can beused without reliance on such a framework. The membrane screen shouldalso be flexible so that it can conform to the column bed. Thisflexibility is advantageous ins the packing process as it allows themembrane screen to conform to the bed of extraction media, resulting ina reduction in dead volume.

Preferably the membrane is a woven or non-woven mesh of fibers that maybe a mesh weave, a random orientated mat of fibers i.e. a “ ” polymerpaper,” a spun bonded mesh, an etched or “pore drilled” paper ormembrane such as nuclear track etched membrane or an electrolytic mesh(see, e.g., U.S. Pat. No. 5,556,598). The membrane may be polymer,glass, or metal provided the membrane is low dead volume, allowsmovement of the various sample and processing liquids through the columnbed, may be attached to the column body, is strong enough to withstandthe bed packing process, is strong enough to hold the column bed ofbeads, and does not interfere with the extraction process i.e. does notadsorb or denature the sample molecules.

The frit can be attached to the column body by any means which resultsin a stable attachment. For example, the screen can be bonded to thecolumn body through welding or gluing. Gluing can be done with anysuitable glue, e.g., silicone, cyanoacrylate glue, epoxy glue, and thelike. The glue or weld joint must have the strength required towithstand the process of packing the bed of extraction media and tocontain the extraction media with the chamber. For glue joints, a glueshould be selected employed that does not adsorb or denature the samplemolecules.

Alternatively, a frit can be attached by means of an annular pip, asdescribed in U.S. Pat. No. 5,833,927. This mode of attachment isparticularly suited to embodiment where the frit is a membrane screen.

The frits of the invention, e.g., a membrane screen, can be made fromany material that has the required physical properties as describedherein. Examples of suitable materials include nylon, polyester,polyamide, polycarbonate, cellulose, polyethylene, nitrocellulose,cellulose acetate, polyvinylidine difluoride, polytetrafluoroethylene(PTFE), polypropylene and glass. A specific example of a membrane screenis the 43 μm pore size Spectra/Mesh® polyester mesh material which isavailable from Spectrum Labs (Ranch Dominguez, Calif., PN 145837).

Extraction Column Assembly

The extraction columns of the invention can be constructed by a varietyof methods using the teaching supplied herein. In some embodiments theextraction column can be constructed by the engagement (i.e.,attachment) of upper and lower tubular members that combine to form theextraction column. Examples of this mode of column construction aredescribed in the Examples and depicted in the figures.

For example, an embodiment of the invention wherein in the two tubularmembers are sections of pipette tips is depicted in FIG. 1 (FIG. 2 is anenlarged view of the open lower end and extraction media chamber of thecolumn). This embodiment is constructed from a frustoconical uppertubular member 2 and a frustoconical lower tubular member 3 engagedtherewith. The engaging end 4 of the upper tubular member has a taperedbore that matches the tapered external surfaced of the engaging end 6 ofthe lower tubular member, the engaging end of the lower tubular memberreceiving the engaging end of the upper tubular member in a telescopingrelation. The tapered bore engages the tapered external surface snuglyso as to form a good seal in the assembled column.

A membrane screen 10 is bonded to and extends across the tip of theengaging end of the upper tubular member and constitutes the upper fritof the extraction column. Another membrane screen 14 is bonded to andextends across the tip of the lower tubular member and constitutes thelower frit of the extraction column. The extraction media chamber 16 isdefined by the membrane screens 10 and 14 and the channel surface 18,and is packed with extraction media 20.

The pore volume of the membrane screens 10 and 14 is low to minimize thedead volume of the column. The sample and desorption solution can passdirectly from the vial or reservoir into the bed of extraction media.The low dead volume permits desorption of the analyte into the smallestpossible desorption volume, thereby maximizing analyte concentration.

The volume of the extraction media chamber 16 is variable and can beadjusted by changing the depth to which the upper tubular memberengaging end extends into the lower tubular member, as determined by therelative dimensions of the tapered bore and tapered external surface.

The sealing of the upper tubular member to the lower tubular in thisembodiment is achieved by the friction of a press fit, but couldalternatively be achieved by welding, gluing or similar sealing methods.

FIG. 3 depicts an embodiment of the invention comprising an upper andlower tubular member engaged in a telescoping relation that does notrely on a tapered fit. Instead, in this embodiment the engaging ends 34and 35 are cylindrical, with the outside diameter of 34 matching theinside diameter of 35, so that he concentric engaging end form a snugfit. The engaging ends are sealed through a press fit, welding, gluingor similar sealing methods. The volume of the extraction bed can bevaried by changing how far the length of the engaging end 34 extendsinto engaging end 35. Note that the diameter of the upper tubular member30 is variable, in this case it is wider at the upper open end 31 andtapers down to the narrower engaging end 34. This design allows for alarger volume in the column channel above the extraction media, therebyfacilitating the processing of larger sample volumes and wash volumes.The size and shape of the upper open end can be adapted to conform to apump used in connection with the column. For example, upper open end 31can be tapered outward to form a better friction fit with a pump such asa pipettor or syringe.

A membrane screen 40 is bonded to and extends across the tip 38 ofengaging end 34 and constitutes the upper frit of the extraction column.Another membrane screen 44 is bonded to and extends across the tip 42 ofthe lower tubular member 36 and constitutes the lower frit of theextraction column. The extraction media chamber 46 is defined by themembrane screens 40 and 44 and the open interior channel of lowertubular member 36, and is packed with extraction media 48.

FIG. 4 is a syringe pump embodiment of the invention with a cylindricalbed of extraction media in the tip, and FIG. 5 is an enlargement of thetop of the syringe pump embodiment of FIG. 4. These figures show a lowdead volume column based on using a disposable syringe and column body.Instead of a pipettor, a disposable syringe is used to pump and containthe sample.

The upper portion of this embodiment constitutes a syringe pump with abarrel 50 into which a plunger 52 is positioned for movement along thecentral axis of the barrel. A manual actuator tab 54 is secured to thetop of the plunger 52. A concentric sealing ring 56 is secured to thelower end of the plunger 52. The outer surface 58 of the concentricsealing ring 56 forms a sealing engagement with the inner surface 60 ofthe barrel 50 so that movement of the plunger 52 and sealing ring 56 upor down in the barrel moves liquid up or down the barrel.

The lower end of the barrel 50 is connected to an inner cylinder 62having a projection 64 for engaging a Luer adapter. The bottom edge 66of the inner cylinder 62 has a membrane screen 68 secured thereto. Theinner cylinder 62 slides in an outer sleeve 70 with a conventional Lueradaptor 72 at its upper end. The lower segment 74 of the outer sleeve 70has a diameter smaller than the upper portion 76, outer sleeve 70forming a ledge 78 positioned for abutment with the lower end 66 andmembrane screen 68. A membrane screen 80 is secured to the lower end 82of the lower segment 74. The extraction media chamber 84 is defined bythe upper and lower membrane screens 68 and 80 and the inner channelsurface of the lower segment 74. The extraction beads 86 are positionedin the extraction media chamber 84. The volume of extraction mediachamber 84 can be adjusted by changing the length of the lower segment74.

Other embodiments of the invention exemplifying different methods ofconstruction are also described in the examples.

Pump

In using the extraction columns of the invention a pump is attached tothe upper open end of the column and used to aspirated and discharge thesample from the column. The pump can take any of a variety of forms, solong as it is capable of generating a negative internal column pressureto aspirate a fluid into the column channel through the open lower end.In some embodiments of the invention the pump is also able to generate apositive internal column pressure to discharge fluid out of the openlower end. Alternatively, other methods can be used for dischargingsolution from the column, e.g., centrifugation.

The pump should be sufficiently strong so as to be able to draw adesired sample solution, wash solution and/or desorption solvent throughthe bed of extraction media.

In some embodiments of the invention the pump is capable of controllingthe volume of fluid aspirated and/or discharged from the column, e.g., apipettor. This allows for the metered intake and outtake of solvents,which facilitates more precise elution volumes to maximize samplerecovery and concentration.

Non-limiting examples of suitable pumps include a pipettor, syringe,peristaltic pump, electrokinetic pump, or an induction based fluidicspump.

III. Methods for Using the Extraction Columns

Extraction columns of the invention should be stored under conditionsthat preserve the integrity of the extraction media. For example,columns containing agarose- or sepharose-based extraction media shouldbe stored under cold conditions (e.g., 4 degrees Celsius) and in thepresence of 0.01 percent sodium azide or 20 percent ethanol.

The sample solution can be any solution containing an analyte ofinterest. The invention is particularly useful for extraction andpurification of biological molecules, hence the sample solution is oftenof biological origin, e.g., a cell lysate. In one embodiment of theinvention the sample solution is a hybridoma cell culture supernatant.

Prior to extraction, a conditioning step may be employed. If analyteextraction is incomplete in a single pass, the sample solution can bepassed back and forth through the media several times. An optional washstep between the extraction and desorption steps can also improve thepurity of the final product. Typically water or a buffer is used for thewash solution. The wash solution is preferably one that will removeunwanted contaminants with a minimal desorption of the analyte ofinterest.

The volume of desorption solvent used can be very small, approximatingthe interstitial volume of the bed of extraction media. In certainembodiments of the invention the amount of desorption solvent used isless than 10-fold greater than the interstitial volume of the bed ofextraction media, more preferably less than 5-fold greater than theinterstitial volume of the bed of extraction media, still morepreferably less than 3-fold greater than the interstitial volume of thebed of extraction media, still more preferably less than 2-fold greaterthan the interstitial volume of the bed of extraction media, and mostpreferably is equal to or less than the interstitial volume of the bedof extraction media.

The desorption solvent will vary depending upon the nature of theanalyte and extraction media. For example, where the analyte is aHis-tagged protein and the extraction media an IMAC resin, thedesorption solution will contain imidazole or the like to release theprotein from the resin. In some cases desorption is achieved by a changein pH or ionic strength, e.g., by using low pH or high ionic strengthdesorption solution. A suitable desorption solution can be arrived atusing available knowledge by one of skill in the art.

In one embodiment, the extraction column may be used formultidimensional stepwise solid phase extraction of isotope-codedaffinity tagged (ICAT) peptides. The fractions are collected on thebasis of increasing ionic strength or pH, and can be processed in theaffinity separation dimension described below, but with suitableadjustments being made for larger sample volumes being introduced intothe affinity capillary and/or possible differences in pH. In certaininstances the fractions collected from the avidin affinity column may beprocessed further for cleavage of the affinity tag from theisotope-coding region, prior to separation in the reversed-phaseseparation dimension described below.

The cleavage can be performed directly upon the collected fraction byphotocleavage as described in Huilin Zhou, et al., Nature Biotech.,19:512 (2002), or acid cleavage with TFA-triethylsilane as described inBrian Williamson, et al., Proceedings of the 50^(th) ASMS Conference onMass Spectrometry and Allied Topics, Orlando, Fla., Jun. 2-6, 2002,Orlando, Fla., Poster # WPA023, or by evaporating the collected fractionto dryness by standard means and adding TFA-triethylsilane reagent toachieve acid cleavage as described in Williamson, et al, 50^(th) ASMSConference Proceedings, Jun. 2-6, 2002, Orlando, Fla., Poster # WPA023(2002).

In instances where the peptide mixture generated by the release,labeling and proteolysis is not excessively complex, it may be possibleto bypass the ion-exchange separation dimension and proceed directly tothe affinity separation dimension. An example of bypassing theion-exchange separation dimension is given in LC Packings/Dionex'Application Note, “2D Analysis of Isotope Coded Affinity Tag (ICAT)Labeled Proteins,” Application Note UltiMate Capillary and Nano LCSystem, Proteomics #09. However, if this strategy is applied it isadvised that some suitable means be applied for removal of theunincorporated ICAT tags prior to introducing the sample to themonomeric avidin column, which would otherwise be removed in theion-exchange separation dimension.

In certain instances it may be possible to bypass the ion-exchangeseparation and affinity separation dimensions and proceed directly fromthe sample protein release, lysis and labeling step (i.e. the first stepdescribed at the beginning of this example) to the reversed-phaseseparation dimension, such as when solid-phase isotope-coded taggingreagents are being utilized as described in Huilin Zhou, et al., NatureBiotech., 19:512 (2002); in this case the cleavage of the isotope-codedpeptide from the solid-phase support can be achieved by photocleavage asdescribed in Huilin Zhou, et al., Nature Biotech., 19:512 (2002) or byacid cleavage as described in Brian Williamson, et al., Proceedings ofthe 50^(th) ASMS Conference on Mass Spectrometry and Allied Topics,Orlando, Fla., Jun. 2-6, 2002, Orlando, Fla., Poster # WPA023.

The device, apparatus and method of this invention can be used toprepare materials for protein chips, DNA chips or other biochips.

Protein chips dynamics can be represented by the following equation:

A+B=AB

AB is capable of generating an analytical signal, where A is thechip-bound moiety and B is its cognate binder introduced to the chip. Anassumption of specific interactions is always assumed. Binding eventsother than “AB” can have the appearance of AB, the variance being causedby non-A (i.e. contaminating) moieties having some affinity for B, non-B(i.e. contaminating) moieties having some affinity for A, or acombinations of the two; any of these events will have the appearance ofa true AB event. This characteristic will define the success or failureof a particular protein chip experiment, and is the most trivialized orignored aspects of the technology.

For some non-protein chips (specifically DNA chips), the A groups do notrequire purification or enrichment since they are synthesized in place,or are amplified via PCR and spotted. With the exception of very shortpeptides, the structural complexity of proteins will not allow foron-chip synthesis of A. Therefore, preparation of A materials for usewithin protein chips will place a premium on the purity of the material.In addition, the A materials will often need to be highly enriched so asto provide maximum opportunity for AB to occur.

Protein chips are characterized by having small volumes of “A” appliedto the surface. The volumes are often on the order of 10 mL or less foreach spot. Since many proteins are difficult and/or expensive toprepare, the ability to purify and enrich at scales on par with thespots would significantly reduce waste. It would also allow for“just-in-time” purification, so that the chip is prepared just as theprotein is being purified.

Different materials are brought to the chip as A, and each materialrequire purification and/or enrichment. Examples of these materials areantibodies (i.e. IgG, IgY, etc) as affinity molecules, general affinityproteins (i.e. scFvs, Fabs, affibodies, peptides, etc) as affinitymolecules, other proteins that are being screened for general affinitycharacteristics, and nucleic acids/(photo) aptamers as affinitymolecules, for example.

Different means of attaching A to chip surfaces, and each will requirepurification and enrichment procedures that are compatible with theattachment chemistry. Examples of attachment chemistry includedirect/passive immobilization to protein chip substrates, and these canbecome covalent in cases of native thiols associating with goldsurfaces, as one example. Covalent attachment is another method ofattachment of functional groups at chip surface, and these can beself-assembled monolayers with and without additional groups,immobilized hydrogel, and the like. Non-covalent/affinity attachment tofunctional groups/ligands at chip surface is another method ofattachment; examples of this method are ProA or ProG for IgGs,phenyl(di)boronic acid with salicylhydroxamic acid groups; streptavidinmonolayers with biotinylation of native lysines/cysteines, and the like.

The samples or analyte to be brought to the chip can be varied incomposition and mode of interaction with A.

There is more than one way to achieve specific AB interactions throughthe manipulation of B. One means is to remove potentially interferingnon-B contaminants by their specific removal, provided thesecontaminants are sufficiently well-defined such as albumin, fibrin, etc.

Another means is the removal of non-B contaminants by trapping B (eitherindividually or as a class), removing contaminants by washing, andreleasing B. This simultaneously allows for enrichment of B, thusenhancing the sensitivity for the AB event.

Just as the scale of the chip is very small, there are opportunities tomake the scale of the sample small—therefore allowing for analysis ofvery small samples. Since samples are precious materials, the scale ofpurification and enrichment would allow for this to occur. As with chippreparation, this can occur in a “just-in-time” manner.

The detection event requires some manner of A interacting with B, so thecentral player in the detection event (since it isn't part of theprotein chip itself) is B. The means of detecting the presence of B (or,B-like substances described above) are varied and can include label-freedetection of B (or B-like substances) interacting with A such as surfaceplasmon resonance imaging as practiced by HTS Biosystems—grating-coupledSPR or BiaCore—prism or Kretschmann-based SPR, or Micro-cantileverdetection schemes as practiced by Protiveris.

The detection means can include physical labeling of B (or B-likesubstances) interacting with A, followed by spatial imaging of AB pair(i.e. Cy3/Cy5 differential labeling with standard fluorescent imaging aspracticed by BD Biosciences Clontech, radioactive ATP labeling of kinasesubstrates with autoradiography imaging as practiced by Jerini or othersuitable imaging techniques. In the case of fluorescent tagging, one canachieve higher sensitivity with fluorescent waveguide imaging aspracticed by ZeptoSens.

The detection means can also include interaction of AB complex with athird B-specific affinity partner C, where C is capable of generating asignal by being fluorescently tagged, or is tagged with a group thatallows a chemical reaction to occur at that location (such as generationof a fluorescent moiety, direct generation of light, etc). Detection ofthis AB-C binding event can occur via fluorescent imaging as practicedby Zyomyx and SomaLogic, chemilumine-scence imaging as practiced by HTSBiosystems and Hypromatrix, fluorescent imaging via waveguidetechnology, or other suitable detection means.

Arrayers are instruments for spotting nucleic acids, proteins or otherreagent onto chips that are used for molecular biology research ordiagnostic work. The arrayers can be used both in the manufacture of thechips and in the use of the chip. In manufacturing, an arrayer can beused to transport the chemical reactants to specific spots on the chip.This may be a multi-step process as the chemical complex used fordetection is built at each particular spot in the array.

Each process can require sample preparation. In some cases, DNA ispurified and deposited to a surface on a chip. Then samples containingcomplementary DNA or RNA are reacted with the chip. Before the samplescan be reacted, the nucleic acid is purified away from the othermaterials (proteins, particulate, carbohydrates, etc.) found in thesamples. In other cases, protein chips may be manufactured by depositingspecific proteins in an array. Then samples containing proteins can bereacted with various array sites to measure protein/proteininteractions.

In application of mass spectrometry for the analysis of biomolecules,the molecules must be transferred from the liquid or solid phases to gasphase and to vacuum phase. Since most biomolecules are both large andfragile, the most effective methods for their transfer to the vacuumphase are matrix-assisted laser desorption ionization (MALDI) orelectrospray ionization (ESI).

Mass spectrometry provides essentially two methods for analyzingproteins: bottom up and top down analysis. In bottom up analysis, theprotein is manipulated and broken up in a controlled manner (usuallythrough an enzymatic digestion process), analyzed, and then reassembledusing the data from the various parts. Top down analysis works with thewhole protein, optionally using an ion source to break apart the proteinand determine the identity of the protein.

While both methods may require long mass spectrometer analysis times,top down approaches usually require the longest time. Under ideal cases,a static sample is measured and parameters on the manner in which thesource is directed or implemented. The methods in which the data areanalyzed are varied to perform a full analysis of the protein.

Many sample introduction methods introduce samples “on-the-fly.” Thesample is introduced from an HPLC column as continuous flow into thenozzle of the electrospray ionization (ESI) source. In order tointroduce samples so that top down analysis can be implemented, the flowof the sample may be slowed. The method is called peak parking. In thisway, the sample residence time can be increased by a factor of 10 orgreater increasing the sensitivity of the analysis by a factor of 8 orgreater. However, this method is still inflexible and inadequate becausethe analysis must still be performed quickly—often more quickly than theinstrument is capable of performing.

This is also true for introduction of samples from a solid phaseextraction device. One may introduce the entire sample before theanalysis is completed. It is much better to introduce a discrete uniformsample into the mass spectrometer. In this way, the mass spectrometrymethod and procedure can be adapted to the sample in the best manner.

This can be accomplished by using an apparatus where the desorbedmaterial from an open tube extraction device is deposited directly intoan electrospray nozzle.

MALDI is commonly interfaced to time of flight (TOF) mass spectrometers(MALDI-TOF) and ESI is interfaced to quadrupole, ion trap and TOF massanalyzers. Both MALDI and ESI approaches are useful for determining thefull masses of proteins and peptides in mixtures, before and afterpurification and to induce fragmentation of peptides for ms/ms analysis.Modern mass spectrometry is accurate enough to be useful for evaluatingthe correct translation or chemical synthesis of biomolecules. Anydeviation of the observed mass of the sample from its calculated massindicates incorrect synthesis or the presence of post-translational orchemical modifications. Biomolecules can be purposely fragmented in themass spectrometer and the masses of the resulting fragments can beaccurately determined. The patterns of such fragment masses are usefulfor ms/ms sequencing of the peptides and their identification in thedata banks.

Electrospray is performed by mixing the sample with volatile acid andorganic solvent and infusing it through a conductive needle charged withhigh voltage. The charged droplets that are sprayed (or ejected) fromthe needle end, are directed into the mass spectrometer, and are driedup by heat and vacuum as they fly in. After the drops dry, the remainingcharged molecules are directed by electromagnetic lenses into the massdetector and mass analyzed. Electrospray mass spectrometry can be usedto determine the masses of different molecules, from small peptides tointact large proteins. Even though the mass-range of the currentlyavailable instruments is only 2000 to 10000 mass unit, most proteinsbecome multi-charged during the electrospray step and since theinstrument measures the mass to charge ratio (m/z) of the molecules,most proteins are sufficiently charged to have an m/z that is within themass range. To calculate the full mass of the protein from the differentm/z measured, a deconvolution is performed, returning the full mass ofthe proteins.

For MALDI-TOF the proteins are deposited on metal targets, asco-crystallized with an organic matrix. The samples are dried andinserted into the mass spectrometer. After vacuum is established, thematrix crystals absorb the light energy from short flashes of ahigh-energy laser. The matrix rapidly sublimes, carrying with it thebiomolecule into the vacuum phase. The sample and matrix plume enter astrong electromagnetic field that accelerate the charged molecules intoa free flight zone where they fly until they hit a detector located atits far end. The mass of the protein can be calculated from its flighttime. Accurate determination of the masses is obtained by the flighttime to that of a standard of known mass. The flight time isproportional to the log of mass of the protein and the larger proteinsfly slower and reach the detector later.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples, whichare provided by way of illustration, and are not intended to be limitingof the present invention, unless so specified.

EXAMPLES

The following preparations and examples are given to enable thoseskilled in the art to more clearly understand and practice the presentinvention. They should not be construed as limiting the scope of theinvention, but merely as being illustrative and representative thereof.

Example 1 Preparation of an Extraction Column Body from Pipette Tips

Two 1000 μL polypropylene pipette tips of the design shown in FIG. 6(VWR, Brisbane, Calif., PN 53508-987) were used to construct oneextraction column. In this example, two extraction columns wereconstructed: a 10 μL bed volume and 20 μL bed volume. To construct acolumn, various components were made by inserting the tips into severalcustom aluminum cutting tools and cutting the excess material extendingout of the tool with a razor blade to give specified column lengths anddiameters.

Referring to FIG. 7, the first cut 92 was made to the tip 92 of apipette tube 90 to form a 1.25 mm inside diameter hole 94 on the lowercolumn body, and a second cut 96 was made to form a lower column bodysegment 98 having a length of 15.0 mm.

Referring to FIG. 8, a cut 102 was made to the separate pipette tip 100to form the upper column body 104. To make a 10 μL bed volume column,the cut 102 was made to provide a tip 106 outside diameter of 2.09 mm sothat when the upper body was inserted into the lower body, the columnheight of the solid phase media bed 114 (FIG. 10) was 4.5 mm. To make a20 μL bed volume column, the cut 102 was made to provide a tip outsidediameter of 2.55 mm cut so that when the upper body was inserted intothe lower body, the column height of the solid phase media bed 114 (FIG.10) was 7.0 mm.

Referring to FIG. 9, a 43 μm pore size Spectra/Mesh® polyester meshmaterial (Spectrum Labs, Ranch Dominguez, Calif., PN 145837) was cutinto discs by a circular cutting tool (Pace Punches, Inc., Irvine,Calif.) and attached to the ends 106 and 108 upper column and lowercolumn bodies to form the membrane screens 110 and 112. The membranescreens were attached using PLASTIX® cyanoacrylate glue (Loctite, Inc.,Avon, Ohio). The glue was applied to the polypropylene body and thenpressed onto the membrane screen material. Using a razor blade, excessmesh material was removed around the outside perimeter of each columnbody end.

Referring to FIG. 10, the upper column body 104 is inserted into the topof the lower column body segment 98 and pressed downward to compact thesolid phase media bed 114 to eliminate excess dead volume above the topof the bed.

Example 2 Preparation of SEPHAROSE™ Protein G and MEP HYPERCEL™Extraction Columns

Referring to FIG. 9, a suspension of Protein G SEPHAROSE™ 4 Fast Flow,45-165 μm particle size, (Amersham Biosciences, Piscataway, N.J., PN17-0618-01) in water/ethanol was prepared, and an appropriate amount ofmaterial 114 was pipetted into the lower column body 98.

Referring to FIG. 10, the upper column body 104 was pushed into thelower column body 98 so that no dead space was left at the top of thebed 114, that is, at the top of the column bed. Care was taken so that aseal was formed between the upper and lower column bodies 104 and 98while retaining the integrity of the membrane screen bonding to thecolumn bodies.

Several tips of 10 μL and 20 μL bed volumes were prepared. Several MEP(Mercapto-Ethyl-Pyridine) HYPERCEL™ (Ciphergen, Fremont, Calif., PN12035-010) extraction columns were prepared using the same procedure.MEP HyperCel™ resin is a sorbent, 80-100 μm particle size, designed forthe capture and purification of monoclonal and polyclonal antibodies.The extraction columns were stored with an aqueous solution of 0.01%sodium azide in a refrigerator before use.

Example 3 Purification of Anti-Leptin Monoclonal Antibody IgG with 10 μLand 20 μL Bed Volume Protein G SEPHAROSE™ Extraction Columns

A Protein G SEPHAROSE™ 4 Fast Flow (Amersham Biosciences, Piscataway,N.J., PN 17-0618-01) extraction column was prepared as described inExample 2.

Five hundred μL serum-free media (HTS Biosystems, Hopkinton, Mass.)containing IgG (HTS Biosystems, Hopkinton, Mass.) of interest wascombined with 500 μL standard PBS buffer. The resulting 1 mL sample waspulled into the pipette tip, through the Protein G packed bed at a flowrate of approximately 1 mL/min) or roughly 15 cm/min). The sample wasthen pushed out to waste at the same approximate flow rate. Extraneousbuffer was removed from the bed by pulling 1 mL of deionized water intothe pipette column at about 1 mL/min and pushing it out at about 1mL/min. The water was pushed out as much as possible to achieve as dryof a column bed as is possible. The IgG was eluted from the column bedby drawing up an appropriate eluent volume of 100 mM glycine-HCl, pH 2.5(20 μL eluent in the case of a 20 μL bed volume, 15 μL eluent in thecase of a 10 μL bed volume). When the eluent was fully drawn into thebed, it was “pumped” back and forth through the bed five or six times,and the IgG-containing eluent was then fully expelled from the bed. Theeluted material was then neutralized with 100 mM NaH₂PO₄/100 mM Na₂HPO₄(5 μL neutralization buffer in the case of a 20 μL bed volume, 4 μLneutralization buffer in the case of a 10 μL bed volume). The purifiedand enriched antibodies were then ready for arraying.

Example 4 Purification of Anti-Leptin Monoclonal Antibody IgG with 10 μLand 20 μL Bed Volume Protein G SEPHAROSE™ Extraction Columns

A Protein G SEPHAROSE™ 4 Fast Flow (Amersham Biosciences, Piscataway,N.J., PN 17-0618-01) extraction column was prepared as described inExample 2.

Five hundred μL serum-free media (HTS Biosystems, Hopkinton, Mass.)containing IgG (HTS Biosystems, Hopkinton, Mass.) of interest wascombined with 500 μL standard PBS buffer. The resulting 1 mL sample waspulled into the pipette tip, through the Protein G packed bed at a flowrate of approximately 1 mL/min (or roughly 150 cm/min linear velocity).The sample was then pushed out to waste at the same approximate flowrate. Extraneous buffer was removed form the bed by pulling 1 mL ofdeionized water into the pipette column at about 1 mL/min and pushing itout at about 1 mL/min. The water was pushed out as much as possible toachieve as dry of a column bed as is possible. The IgG was eluted fromthe column bed by drawing up an appropriate eluent volume of 10 mMphosphoric acid (H₃PO₄), pH 2.5 (20 μL eluent in the case of a 20 μL bedvolume, 15 μL eluent in the case of a 10 μL bed volume). When the eluentwas fully drawn into the bed, it was “pumped” back and forth through thebed five or six times, and the IgG-containing eluent is then fullyexpelled from the bed. The eluted material was then neutralized with aspecially designed phosphate neutralizing buffer of 100 mM H₂NaPO₄/100mM HNa₂PO₄, pH 7.5 (5 μL neutralization buffer in the case of a 20 μLbed volume, 4 μL neutralization buffer in the case of a 10 μL bedvolume). The purified and enriched antibodies were then ready forarraying.

Example 5 Analysis of Purified IgG with Grating-Coupled Surface PlasmonResonance (GC-SPR)

The anti-leptin monoclonal antibody IgG purified sample from Example 4was analyzed with GC-SPR. The system used for analysis was a FLEX CHIP™Kinetic Analysis System (HTS Biosystems, Hopkinton, Mass.), whichconsists of a plastic optical grating coated with a thin layer of goldon to which an array of biomolecules is immobilized. To immobilize thepurified IgG, the gold-coated grating was cleaned thoroughly with EtOH(10-20 seconds under a stream of EtOH). The gold-coated grating was thenimmersed in a 1 mM solution of 11-mercaptoundecanoic acid (MUA) in EtOHfor 1 hour to allow for the formation of a self-assembled monolayer. Thesurface was rinsed thoroughly with EtOH and ultra-pure water, and driedunder a stream of nitrogen. A fresh solution of 75 mM EDC(1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide hydrochloride) and 15 mMSulfo-NHS(N-Hydroxysulfo-succinimide) was prepared in water. An aliquotof the EDC/NHS solution was delivered to the surface and allowed toreact for 20-30 minutes, and the surface was then rinsed thoroughly withultra-pure water. An aliquot of 1 mg/mL Protein A/G in PBS, pH 7.4 wasdelivered to the surface. The surface was placed in a humid environmentand allowed to react for 1-2 hours. The surface was allowed to air dry,was rinsed with ultra-pure water and then dried under a stream ofnitrogen. Immediately prior to arraying of the IgGs, the surface wasrehydrated by placing in a humidified chamber, such as available withcommercial arraying systems (e.g. Cartesian MicroSys synQUAD System).The purified anti-leptin IgG was arrayed onto the surface as describedpreviously (J. Brockman, et al, “Grating-Coupled SPR: A Platform forRapid, Label-free, Array-Based Affinity Screening of Fabs and Mabs”,12^(th) Annual Antibody Engineering Conference, Dec. 2-6, 2001, SanDiego, Calif.) and the surface was introduced to the HTS Biosystems FLEXCHIP System. 150 nM leptin in PBS, pH 7.4 was introduced to the surfacethrough the FLEX CHIP System, and real-time binding signals werecollected as described previously (ibid.). These real-time bindingsignals were mathematically processed in a manner described previously(D. Myszka, “Kinetic analysis of macromolecular interactions usingsurface plasmon resonance biosensors”, Current Opinion in Biotechnology,1997, Vol 8, pp. 50-57) for extraction of the association rate (k_(a)),dissociation rate (k_(d)), and the dissociation affinity constant(K_(d)=k_(d)/k_(a)). The kinetic data obtained is shown in Table IIbelow.

TABLE II Serum-free medium PBS No processing Mean K_(d) 18 nM 3.2 nM(Adequate [IgG]) Starting 500 μg/mL 500 μg/mL [IgG] With processing MeanK_(d) 6.6 nM 5.9 nM* (Insufficient [IgG] Starting 20 μg/mL 500 μg/mL*[IgG] *500 μg/mL IgG in PBS was not processed, but was included in theSPR analysis for the purpose of comparing dissociation affinityconstants calculated for each

The first set of data for “No processing” indicates that when sufficientIgG is present for detection (500 μg/mL) that the constituents from theserum-free medium can contribute to inaccuracies. These data indicatefor equal concentrations of IgG spotted within an experiment, thecalculated dissociation affinity constant can be nearly six-folddifferent from one another (18 nM vs. 3.2 nM). This can only be a resultof components within the serum-free medium being co-arrayed with theIgG, since the concentration and composition of IgG is identical foreach sample. Therefore, there is a demonstrated need for removal of anyextraneous components prior to arraying, which is independent of IgGconcentration.

The second set of data for “With processing” indicates that wheninsufficient IgG quantities are present for detection (20 μg/mL) thatsample processing not only allows for generation of sufficientprocessable signals, but also eliminates the inaccuracies generated fromextraneous components. These data indicate that the dissociationaffinity constants are virtually identical for 500 μg/mL purified IgG inPBS (unprocessed) as those calculated from 20 μg/mL IgG in serum-freemedium once processed with the current invention (5.9 nM vs. 6.6 nM).

Example 6 Purification of Nucleic Acids with an Extraction Column

Columns from Example 1 are bonded with a 21 μm pore size SPECTRA/MESH®polyester mesh material (Spectrum Labs, Ranch Dominguez, Calif., PN148244) by the same procedure as described in Example 2. A 10 μL bedvolume column is filled with PELLICULAR C18 (Alltech, Deerfield, Ill.,PN 28551), particle size 30-50 μm. One end of the extraction column isconnected to a pipettor pump (Gilson, Middleton, Wis., P-1000PipetteMan) and the other end is movable and is connected to anapparatus where the materials may be taken up or deposited at differentlocations.

The extraction column consists of a 1 mL syringe (VWR, Brisbane, Calif.,PN 53548-000), with one end connected to a pipettor pump (Gilson,Middleton, Wis., P-1000 PipetteMan) and the other end is movable and isconnected to an apparatus where the materials may be taken up ordeposited at different locations.

A 100 μL sample containing 0.01 μg of DNA is prepared using PCRamplification of a 110 bp sequence spanning the allelic MstII site inthe human hemoglobin gene according to the procedure described in U.S.Pat. No. 4,683,195. A 10 μL concentrate of triethylammonium acetate(TEAA) is added so that the final volume of the solution is 110 μL andthe concentration of the TEAA in the sample is 100 mM. The sample isintroduced into the column and the DNA/TEAA ion pair complex isadsorbed.

The sample is blown out of the column and 10 μL of 50% (v/v)acetonitrile/water is passed through the column, desorbing the DNA, andthe sample is deposited into a vial for analysis.

Example 7 Desalting Proteins with an Extraction Column

Columns from Example 1 are bonded with a 21 μm pore size SPECTRA/MESH®polyester mesh material (Spectrum Labs, Ranch Dominguez, Calif., PN148244) by the same procedure as described in Example 2. A 10 μL bedvolume column is filled with PELLICULAR C18 (Alltech, Deerfield, Ill.,PN 28551), particle size 30-50 μm. One end of the extraction column isconnected to a pipettor pump (Gilson, Middleton, Wis., P-1000PipetteMan) and the other end is movable and is connected to anapparatus where the materials may be taken up or deposited at differentlocations.

The sample is a 100 μL solution containing 0.1 μg of Protein kinase A ina phosphate buffer saline (0.9% w/v NaCl, 10 mM sodium phosphate, pH7.2) (PBS) buffer. Ten μL of 10% aqueous solution of trifluoroaceticacid (TFA) is added so that the final volume of the solution is 110 μLand the concentration of the TFA in the sample is 0.1%. The sample isintroduced into the column and the protein/TFA complex is adsorbed tothe reverse phase of the bed.

The sample is blown out of the column and 10 μL of 50% (v/v)acetonitrile/water is passed through the column, desorbing the proteinfrom the bed of extraction media, and the sample is deposited into avial for analysis.

Alternatively, the bed may be washed with 10 μL of aqueous 0.1% TFA.This solution is ejected from the column and the protein is desorbed anddeposited into the vile.

If necessary, alternatively 1% heptafluorobutyric acid (HFBA) is usedinstead of TFA to reduce ion suppression effect when the sample isanalyzed by electrospray ion trap mass spectrometry.

Example 8 Straight Connection Configuration

This example describes an embodiment wherein the column body isconstructed by engaging upper tubular members and membrane screens in astraight configuration.

Referring to FIG. 11, the column consists of an upper tubular member120, a lower tubular member 122, a top membrane screen 124, a bottommembrane screen 126, and a lower tubular circle 134 to hold the bottommembrane screen in place. The top membrane screen is held in place bythe upper and lower tubular members. The top membrane screen, bottommembrane screen and the channel surface 130 of the lower tubular memberdefine an extraction media chamber 128, which contains a bed ofextraction media 132 (i.e., packing material). The tubular members asdepicted in FIG. 11 are frustoconical in shape, but in relatedembodiments could take other shapes, e.g, cyclindrical.

To construct a column, various components are made by forming injectedmolded members from polypropylene or machined members from PEEK polymerto give specified column lengths and diameters and ends that can fittogether, i.e., engage with one another. The configuration of the maleand female portions of the column body is shaped differently dependingon the method used to assemble the parts and the method used to keep theparts together.

The components are glued or welded. Alternatively, they are snappedtogether. In the case of snapping the pieces together, the femaleportion contains a lip and the male portion contains a ridge that willhold and seal the pieces once they are assembled. The membrane screen iseither cut automatically during the assembly process or is trimmed afterassembly.

Example 9 End Cap and Retainer Ring Configuration

This example describes an embodiment where an end cap and retainer ringconfiguration is used to retain the membrane screens containing a 20 μlbed of column packing material. The embodiment is depicted in FIG. 12.

Referring to the figure, pipette tip 140 (VWR, Brisbane, Calif., PN53508-987) was cut with a razor blade to have a flat and straight bottomend 142 with the smooth sides such that a press fit can be performedlater. An end cap 144 was machined from PEEK polymer tubing to containthe bottom membrane screen 146.

Two different diameter screens were cut from polyester mesh (SpectrumLabs, Ranch Dominguez, Calif., PN 145836) by a circular cutting tool(Pace Punches, Inc., Irving, Calif.), one for the top membrane screen148 and the other for the bottom membrane screen 146. The bottommembrane screen was placed into the end cap and pressed onto the end ofthe cut pipette tip.

A 20 μL volume bed of beads 150 was formed by pipetting a 40 μL of 50%slurry of protein G agarose resin into the column body.

Two retainer rings were used to hold the membrane screen in place on topof the bed of beads. The retainer rings were prepared by taking ⅛ inchdiameter polypropylene tubing and cutting thin circles from the tubingwith a razor blade. A first retainer ring 152 was placed into the columnand pushed down to the top of the bed with a metal rod of similardiameter. The membrane screen 148 was placed on top of the firstretainer ring and then a second retainer ring 154 was pushed down to“sandwich” the membrane screen while at the same time pushing the wholescreen configuration to the top of the bed and ensuring that all deadvolume was removed. The membrane is flexible and naturally forms itselfto the top of the bed.

The column was connected to a 1000 μL pipettor (Gilson, Middleton, Wis.,P-1000 PipetteMan) and water was pumped through the bed and dispensedfrom the bed. The column had low resistance to flow for water solvent.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover and variations,uses, or adaptations of the invention that follow, in general, theprinciples of the invention, including such departures from the presentdisclosure as come within known or customary practice within the art towhich the invention pertains and as may be applied to the essentialfeatures hereinbefore set forth. Moreover, the fact that certain aspectsof the invention are pointed out as preferred embodiments is notintended to in any way limit the invention to such preferredembodiments.

1. A pipette tip extraction column comprising: i) a column body havingan open upper end for attachment to a pump, an open lower end forpassing fluid into and out of the column body, and an open channelbetween the upper and lower end of the column body, wherein the columnbody comprises a pipette tip; ii) a bottom frit bonded to and extendingacross the open channel, the bottom frit having a pore volume, andwherein the bottom frit is located at or near the open lower end of thecolumn body; iii) a top frit bonded to and extending across the openchannel between the bottom frit and the open upper end of the columnbody, wherein the top frit and bottom frit are flexible, and wherein thetop frit, bottom frit, and channel surface define an extraction mediachamber; and iv) a bed of gel resin extraction media positioned insidethe extraction media chamber.
 2. The pipette tip extraction column ofclaim 1, wherein the top frit or the bottom frit is a membrane screen.3. The pipette tip extraction column of claim 1, wherein the gel resinis selected from the group consisting of agarose and sepharose.
 4. Thepipette tip extraction column of claim 1, wherein the gel resinextraction media comprises an affinity binding group having an affinityfor a biological molecule of interest.
 5. The pipette tip extractioncolumn of claim 4, wherein the affinity binding group is selected fromthe group consisting of Protein A, Protein G, Protein L and animmobilized metal.
 6. The pipette tip extraction column of claim 1,wherein the upper end of the column body is attached to a pump foraspirating fluid through the lower end of the column body.
 7. Thepipette tip extraction column of claim 6, wherein the pump is a pipettoror a syringe.
 8. A pipette tip extraction column comprising: i) a columnbody having an open upper end for attachment to a pump, an open lowerend for passing fluid into and out of the column body, and an openchannel between the upper and lower end of the column body, wherein thecolumn body comprises a pipette tip; ii) a bottom frit bonded to andextending across the open channel, the bottom frit having a pore volume,and wherein the bottom frit is located at or near the open lower end ofthe column body; iii) a top frit bonded to and extending across the openchannel between the bottom frit and the open upper end of the columnbody, wherein the top frit and bottom frit are flexible membranescreens, and wherein the top frit and the bottom frit are bonded to thecolumn body by gluing or welding, and wherein the top frit, bottom frit,and channel surface define an extraction media chamber; and iv) a bed ofgel resin extraction media positioned inside the extraction mediachamber.
 9. The pipette tip extraction column of claim 8, wherein thegel resin is selected from the group consisting of agarose andsepharose.
 10. The pipette tip extraction column of claim 8, wherein thegel resin extraction media comprises an affinity binding group having anaffinity for a biological molecule of interest.
 11. The pipette tipextraction column of claim 10, wherein the affinity binding group isselected from the group consisting of Protein A, Protein G, Protein Land an immobilized metal.
 12. The pipette tip extraction column of claim8, wherein the upper end of the column body is attached to a pump foraspirating fluid through the lower end of the column body.
 13. Thepipette tip extraction column of claim 12, wherein the pump is apipettor or a syringe.
 14. A pipette tip extraction column comprising:i) a column body having an open upper end for attachment to a pump, anopen lower end for passing fluid into and out of the column body, and anopen channel between the upper and lower end of the column body, whereinthe column body comprises a pipette tip; ii) a bottom frit extendingacross the open channel, the bottom frit having a pore volume, andwherein the bottom frit is located at or near the open lower end of thecolumn body; iii) a top frit extending across the open channel betweenthe bottom frit and the open upper end of the column body, wherein thetop frit and bottom frit are attached to the column body by a meansselected from the group consisting of an annular pip, gluing, andwelding, and wherein the top frit, bottom frit, and channel surfacedefine an extraction media chamber; and iv) a bed of gel resinextraction media positioned inside the extraction media chamber.
 15. Thepipette tip extraction column of claim 14, wherein the top frit or thebottom frit is a membrane screen.
 16. The pipette tip extraction columnof claim 14, wherein the gel resin is selected from the group consistingof agarose and sepharose.
 17. The pipette tip extraction column of claim14, wherein the gel resin extraction media comprises an affinity bindinggroup having an affinity for a biological molecule of interest.
 18. Thepipette tip extraction column of claim 17, wherein the affinity bindinggroup is selected from the group consisting of Protein A, Protein G,Protein L and an immobilized metal.
 19. The pipette tip extractioncolumn of claim 14, wherein the upper end of the column body is attachedto a pump for aspirating fluid through the lower end of the column body.20. The pipette tip extraction column of claim 19, wherein the pump is apipettor or a syringe.